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Purpose:In this first-in-human, phase I, GVHD prevention trial (NCT02891603), we combine pacritinib (PAC), a JAK2 inhibitor, with sirolimus to concurrently reduce T-cell costimulation via mTOR and IL6 activity. We evaluate the safety of pacritinib when administered with sirolimus plus low-dose tacrolimus (PAC/SIR/TAC) after allogeneic hematopoietic cell transplantation.Patients and Methods:The preclinical efficacy and immune modulation of PAC/SIR were investigated in xenogeneic GVHD. Our phase I trial followed a 3+3 dose-escalation design, including dose level 1 (pacritinib 100 mg daily), level 2 (pacritinib 100 mg twice daily), and level 3 (pacritinib 200 mg twice daily). The primary endpoint was to identify the lowest biologically active and safe dose of pacritinib with SIR/TAC (n = 12). Acute GVHD was scored through day +100. Allografts included 8/8 HLA-matched related or unrelated donor peripheral blood stem cells.Results:In mice, we show that dual JAK2/mTOR inhibition significantly reduces xenogeneic GVHD and increases peripheral regulatory T cell (Treg) potency as well as Treg induction from conventional CD4+ T cells. Pacritinib 100 mg twice a day was identified as the minimum biologically active and safe dose for further study. JAK2/mTOR inhibition suppresses pathogenic Th1 and Th17 cells, spares Tregs and antileukemia effector cells, and exhibits preliminary activity in preventing GVHD. PAC/SIR/TAC preserves donor cytomegalovirus (CMV) immunity and permits timely engraftment without cytopenias.Conclusions:We demonstrate that PAC/SIR/TAC is safe and preliminarily limits acute GVHD, preserves donor CMV immunity, and permits timely engraftment. The efficacy of PAC/SIR/TAC will be tested in our ongoing phase II GVHD prevention trial.

The table shows maximum acute GVHD organ stage and overall grade observed through day +100 after transplant on the PAC/SIR/TAC phase I trial.

The table shows the characteristics of patients treated on the PAC/SIR/TAC phase I trial.

Impact of JAK2/mTOR blockade on human NK cell function and proliferation. A) Graph shows the cytolytic activity ({plus minus}SEM) of NK cells after 4 hours of culture with K562 target cells, while exposed to DMSO, ruxolitinib (lμM), pacritinib (1.25μM), sirolimus 10ng/ml, or a combination of pacritinib plus sirolimus. B) Human NK cells (105 ) were stimulated with a cytokine cocktail of recombinant human (rh) IL-2 (200 IU/ml) and rhll-15 (10 ng/ml), in the presence of DMSO, ruxolitinib, pacritinib, sirolimus, or pacritinib plus sirolimus. Drugs were added once on day 0. Cytokines were replenished on day +3. Bar graph shows NK cell proliferation ({plus minus}SEM) on day +5 of the culture using a colorimetric assay.

The table shows the pharmacokinetic analysis of pacritinib concentration over time.

The table shows pacritinib adherence during treatment period according to dose level on the PAC/SIR/TAC phase I trial.

Safety measures monitored on trial including (A) QTcF, (B) cardiac ejection fraction, (C) prothrombin time, (D) partial thromboplastin time, (E) and thrombin time

The table shows adverse events per CTCAE version 5 encountered during the PAC/SIR/TAC phase I trial.

CD4+ T cell Aurora kinase A activation as a GVHD resistance mechanism. A) Graph shows frequency of CD4+, pHistone 3 serine 10+ (pH3ser10) (downstream of CD28, and parallel to mTOR) T cells (mean{plus minus} SEM) at day +21 among patients treated on dose level 1 or dose level 2 of the phase I trial compared to baseline control values. B) Graph depicts the frequency (mean {plus minus} SEM) of CD4+, pH3ser10+ T cells recovered from NSG spleens at day +14, following transplantation with human PBMCs and then treated with vehicle, pacritinib, 531-201, sirolimus, pacritinib plus sirolimus, or 531-201 plus sirolimus for 2 weeks.

The table shows the maximum chronic GVHD organ involvement and overall score per NIH Diagnosis and Staging Criteria through full extent of study follow up on the PAC/SIR/TAC phase I trial.

Impact of JAK2 or STAT3 inhibition plus mTOR blockade on DC-allostimulated human T cell production of IFNγ, IL-13, and GM-CSF. 5-day alloMLRs were treated with DMSO vehicle control, pacritinib 1.25μM, S3I-201 5μM, sirolimus 10ng/ml, pacritinib plus sirolimus, or S3I-201 plus sirolimus. The graphs show the concentrations of A) IFNγ, B) IL-13, and C) GM-CSF (mean{plus minus}SEM) as measured in the supernatants of the culture medium by a cytokine multiplex assay.

Extended Methods including information and details regarding flow cytometry, cell lines, allogeneic mixed leukocyte reactions, Treg Suppression Assays, Xenogeneic GVHD modeling, and key reagents.

The table shows donor chimerism in unsorted bone marrow, and peripheral blood CD3 and CD33 populations from patients treated on the PAC/SIR/TAC phase I trial.

Serial measures of absolute neutrophil count (ANC), hemoglobin (Hgb) and platelet counts (PLT) through complete pacritinib treatment period

The table shows donor/recipient CMV serostatus.

Impact of JAK2/mTOR blockade on human T cell responses toward CMV, EBV, Influenza, and tetanus stimulation. A, B) Graphs show the recall proliferation of CD4+ and CD8+ T cell proliferation in response to CEFT peptides. C, D) Graph shows the frequency of IFNy+, CD107a+ CD4+ or cos+ T cells after stimulation with CEFT peptides.

Invasive fungal disease (IFD) is a major source of morbidity and mortality for hematopoietic cell transplant (HCT) recipients. Non-invasive biomarkers, such as the beta-D-glucan assay, may improve the diagnosis of IFD. The objective was to define the utility of surveillance testing using Fungitell® beta-D-glucan (BDG) assay in children receiving antifungal prophylaxis in the immediate post-HCT period.Weekly surveillance blood testing with the Fungitell® BDG assay was performed during the early post-HCT period in the context of a randomized trial of children, adolescents, and young adults undergoing allogeneic HCT allocated to triazole or caspofungin prophylaxis. Positivity was defined at the manufacturer cutoff of 80 pg/ml. IFD was adjudicated using blinded central reviewers. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the Fungitell® BDG assay for the outcome of proven or probable IFD.A total of 51 patients (out of 290 patients in the parent trial) contributed blood specimens. In total, 278 specimens were evaluated. Specificity was 80.8% (95% confidence interval [CI]: 75.6%-85.3%), and NPV was over 99% (95% CI: 86.8%-99.9%). However, there were no true positive results, resulting in sensitivity of 0% (95% CI: 0.0%-84.2%) and PPV of 0% (95% CI: 0.0%-6.7%).Fungitell® BDG screening is of limited utility in diagnosing IFD in the post-HCT period, mainly due to high false-positive rates. Fungitell® BDG surveillance testing should not be performed in children during the early post-HCT period while receiving antifungal prophylaxis as the pretest probability for IFD is low.

Background: The aim was to demonstrate the feasibility and technique of gonadal sparing total body irradiation (TBI) with helical tomotherapy. Total body irradiation is a common part of the conditioning regimen prior to allogeneic stem cell transplantation. Shielding or dose-reduction to the gonads is often desired to preserve fertility, particularly in young patients undergoing transplant for non-malignant indications. Helical tomotherapy (HT) has been shown to be superior to traditional TBI delivery for organ at risk (OAR) doses and dose homogeneity. Materials and methods: We present two representative cases (one male and one female) to illustrate the feasibility of this technique, each of whom received 3Gy in a single fraction prior to allogeneic stem cell transplant for benign indications. The planning target volume (PTV) included the whole body with a subtraction of OARs including the lungs, heart, and brain (each contracted by 1cm) as well as the gonads (testicles expanded by 5 cm and ovaries expanded by 0.5 cm). Results: For the male patient we achieved a homogeneity index of 1.35 with a maximum and median planned dose to the testes of 0.53 Gy and 0.35 Gy, respectively. In-vivo dosimetry demonstrated an actual received dose of 0.48 Gy. For the female patient we achieved a homogeneity index of 1.13 with a maximum and median planned dose to the ovaries of 1.66 Gy and 0.86 Gy, respectively. Conclusion: Gonadal sparing TBI is feasible and deliverable using HT in patients with non-malignant diseases requiring TBI as part of a pre-stem cell transplant conditioning regimen.

This study assessed the efficacy and safety of atropine-midazolam-ketamine as a sedative or analgesic regimen in pediatric oncology patients undergoing painful procedures. Of 136 consecutive pediatric patients undergoing painful procedures from June 1, 1997 to June 30, 1998, 69 patients (39 males and 30 females; mean age, 7.3 ± 4.4 years) underwent 255 procedures using intravenous atropine 0.01 mg/kg plus midazolam 0.05 mg/kg plus ketamine up to 2.0 mg/kg. Sedation monitoring forms for these 69 patients were examined retrospectively. Vital signs, pulse oximetry, patients status, clinical effects, adverse events, and patients or parent comments were recorded at 5-minute intervals from initiation of sedation until the child was awake. Clinical efficacy was defined as procedure completion with minimal patient distress; a clinically significant adverse event was defined as baseline deviation requiring acute medical intervention to prevent or ameliorate clinical deterioration. Transient clinical effects were defined as self-limited deviations in respiratory rate, heart rate, or blood pressure > 15% of age-specific norms. Most of the participants were white males with leukemia or lymphoma who were undergoing lumbar punctures. Twenty-three procedures (9.0%) required additional administration of midazolam or ketamine for pain control or sedation. The 3 most frequent transient effects were hypertension in 71 procedures (27.8%), hypotension in 53 procedures (20.8%), and tachycardia in 49 procedures (19.2%). Twenty-five procedures (9.8%) had associated complications. Ten procedures (3.9%) had ketamine-associated emergent reactions, ranging from laughing and verbosity to screaming and crying. Nine procedures (3.5%) were associated with nausea, vomiting, or dry heaving, and 2 (0.8%) were associated with shaking chills after procedure completion. The clinically significant adverse event rate was 1.6% (n = 4, all during lumbar punctures). In 1 case, the procedure was terminated prematurely because of laryngospasm; in 3 cases oxygen supplementation was required because of hypoxemia. Clinically significant adverse events were associated with younger patients and increased ketamine doses. None of the adverse events had long-term sequelae. Results of the present study suggest that the atrophine-midazolam-ketamine regimen is efficacious and demonstrates an acceptable safety profile in the majority of pediatric oncology patients undergoing properly supervised sedation

The role of SCT in pediatric oncology has continued to evolve with the introduction of new therapeutic agents and immunological insights into cancer. COG has focused its efforts on the study of hematopoietic stem cell transplantation in the treatment of pediatric malignancies in several major multi-institutional Phase II and Phase III studies. These studies include addressing the impact of allogenicity in ALL (ASCT0431), and establishing autologous stem cell transplant as the standard of care in neuroblastoma. Reducing transplant-associated toxicity was addressed in the ASCT0521 study, where the TNFα inhibitor etanercept was tested for the treatment of idiopathic pneumonia syndrome. Impact of cell dose was explored in the single versus tandem umbilical cord blood study CTN-0501, in close collaboration with the BMT-CTN.

Patients with neuroblastoma who present with the syndrome of opsoclonus and myoclonus enjoy a remarkably good prognosis independent of their stage of disease or their age at diagnosis. The presence of N-myc amplification also has been found to be an independent prognostic factor in neuroblastoma. Patients with multicopy N-myc tumors have rapid tumor progression whereas those with single-copy tumors have a significantly better progression-free survival. The authors examined four primary, untreated neuroblastomas for the N-myc copy number from patients who presented with opsoclonus and myoclonus. All four tumors had single copies of N-myc, and all four patients are alive with no evidence of recurrent disease with 6+ to 54+ months' follow-up. This appears to be the only report of N-myc analysis in this group of children. It would be interesting to analyze more neuroblastomas from patients who present with opsoclonus and myoclonus to determine how many of these patients have single N-myc copy tumors.

Clinical research published since the first evidence-based review on the role of hematopoietic stem cell transplantation (SCT) in the treatment of pediatric acute lymphoblastic leukemia (ALL) is presented and critically evaluated in this update. Treatment recommendations are provided by an expert panel. Allogeneic SCT is recommended for children who: are in second complete remission (CR2) after experiencing an early marrow relapse for precursor-B ALL; experienced primary induction failure, but subsequently achieved a CR1; have T-lineage ALL in CR2; or have ALL in third or greater remission. Although the 2005 pediatric ALL evidence-based review (EBR) recommended allogeneic SCT for children with Philadelphia chromosome positive (Ph+) ALL in CR1, preliminary tyrosine kinase inhibitor (TKI) data demonstrate that early outcomes are comparable for allogeneic SCT and chemotherapy + imatinib. Based on the evidence, autologous SCT is not recommended for ALL in CR1. Allogeneic SCT is not recommended for: T-lineage ALL in CR1; mixed-lineage leukemia (MLL)+ ALL when it is the sole adverse risk factor; isolated central nervous system (CNS) relapse in precursor-B ALL. Based on expert opinion, allogeneic SCT may be considered for hypodiploid ALL and persistent minimal residual disease [corrected] (MRD) positivity in ALL in CR1 or greater, although these are areas that need further study. Treatment recommendations pertaining to various transplantation techniques are also provided, as are areas of needed future research.

The advent of the bromodeoxyuridine(BrdU)-differential staining techniques has greatly facilitated the detection of sister chromatid exchanges (SCE). These SCE have been demonstrated to be an accurate reflection of DNA damage both in vitro in cultured cells and in vivo in mouse and rat bone marrow and spleen cells. In this review, we examine the effect of cellular aging on both baseline and mutagen-induced SCE levels. In all systems examined, aging did not appear to significantly affect the baseline levels of SCE. However, in human fibroblast cultures we have found a significant decrease in the levels of mutagen-induced SCE as a function of both in vitro passage level ( in vitro aging) and the age of the cell culture donor ( in vivo aging). In addition we have found a similar decrease in mutagen-induced SCE levels in both mouse and rat bone marrow cells and mouse spleen cells where examinations were performed entirely in vivo . Diminished mutagen-induced SCE levels were obtained with a wide variety of agents including mitomycin-C, cyclophosphamide, adriamycin, ethyl methanesulfonate and N -acetyl-2-acetoxyaminofluorene. These decreased SCE levels were accompanied by increased frequencies of chromosomal aberrations in the older cell populations. If SCE represents a form of DNA repair as has been suggested by several investigators, our findings would indicate impaired DNA repair occurring in old cells.